Purification of Yeast Artificial Chromosome DNA for Microinjection Using Pulsed-Field Gel Electrophoresis and Ultrafiltration

2018 ◽  
Vol 2018 (8) ◽  
pp. pdb.prot093948 ◽  
Author(s):  
Lluis Montoliu
Keyword(s):  
Gel Electrophoresis ◽  
Yeast Artificial Chromosome ◽  
Artificial Chromosome ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field

Construction of a MluI-YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions

Genome ◽  
1997 ◽  
Vol 40 (6) ◽  
pp. 896-902 ◽  
Author(s):  
Michael Kleine ◽  
Christian Jung ◽  
Wolfgang Michalek ◽  
Thomas Diefenthal ◽  
Harald Dargatz
Keyword(s):  
Hordeum Vulgare ◽  
Gel Electrophoresis ◽  
Yeast Artificial Chromosome ◽  
Single Copy ◽  
Pulsed Field Gel Electrophoresis ◽  
Hordeum Vulgare L ◽  
High Molecular Weight Dna ◽  
Pulsed Field ◽  
Artificial Chromosomes ◽  
Yac Library

We describe the construction of a specific yeast artificial chromosome (YAC) library from barley (Hordeum vulgare L.) using the vector pYAC-RC. The library was generated by total digestion of high molecular weight DNA with the infrequently cutting restriction enzyme MluI. Only 10–30% of the colonies were recombinant, as visualized by red–white selection and subsequent pulsed-field gel electrophoresis analysis. About 17 000 individual recombinant YAC clones with insert sizes ranging from 50 to 700 kb, with a mean of 170 kb, were selected. No chloroplast sequences were detected and the proportion of YAC clones containing BARE–1 copia–like retroelements is about 5%. Screening of the library with a single-copy RFLP marker closely linked to the Mla locus yielded three identical clones of the same size. Insert termini of randomly chosen YAC clones were investigated with respect to their redundancy in the barley genome and compared with termini of YAC clones from an EcoRI-based YAC library, resulting in a fourfold enrichment of single-copy sequences at the MluI vector–insert junctions.Key words: yeast artificial chromosomes, YAC, Hordeum vulgare, pulsed-field gel electrophoresis.

A bacterial artificial chromosome based physical map of the Ustilago maydis genome

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 207-216 ◽  
Author(s):  
Khalid Meksem ◽  
Jeffry Shultz ◽  
Faiza Tebbji ◽  
Aziz Jamai ◽  
Jürgen Henrich ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Gel Electrophoresis ◽  
Physical Map ◽  
Artificial Chromosome ◽  
Ustilago Maydis ◽  
Pulsed Field Gel Electrophoresis ◽  
Pulsed Field ◽  
Artificial Chromosomes ◽  
Bac Clones ◽  
Copy Dna

Ustilago maydis, a basidiomycete, is a model organism among phytopathogenic fungi. A physical map of U. maydis strain 521 was developed from bacterial artificial chromosome (BAC) clones. BAC fingerprints used polyacrylamide gel electrophoresis to separate restriction fragments. Fragments were labeled at the HindIII site and codigested with HaeIII to reduce fragments to 50–750 bp. Contiguous overlapping sets of clones (contigs) were assembled at nine stringencies (from P ≤ 1 x 10–6 to 1 x 10–24). Each assembly nucleated contigs with different percentages of bands overlapping between clones (from 20% to 97%). The number of clones per contig decreased linearly from 41 to 12 from P ≤ 1 x 10–7 to 1 x 10–12. The number of separate contigs increased from 56 to 150 over the same range. A hybridization-based physical map of the same BAC clones was compared with the fingerprint contigs built at P ≤ 1 × 10–7. The two methods provided consistent physical maps that were largely validated by genome sequence. The combined hybridization and fingerprint physical map provided a minimum tile path composed of 258 BAC clones (18–20 Mbp) distributed among 28 merged contigs. The genome of U. maydis was estimated to be 20.5 Mbp by pulsed-field gel electrophoresis and 24 Mbp by BAC fingerprints. There were 23 separate chromosomes inferred by both pulsed-field gel electrophoresis and fingerprint contigs. Only 11 of the tile path BAC clones contained recognizable centromere, telomere, and subtelomere repeats (high-copy DNA), suggesting that repeats caused some false merges. There were 247 tile path BAC clones that encompassed about 17.5 Mbp of low-copy DNA sequence. BAC clones are available for repeat and unique gene cluster analysis including tDNA-mediated transformation. Program FingerPrint Contigs maps aligned with each chromosome can be viewed at http://www.siu.edu/~meksem/ustilago_maydis/.Key words: Ustilago maydis, physical map, bacterial artificial chromosomes, whole-genome sequencing.

scholarly journals Use of Pulsed-Field Gel Electrophoresis to Determine the Source of Methicillin-Resistant Staphylococcus aureus Bacteremia

2021 ◽  
Vol 13 (3) ◽  
pp. 602-610
Author(s):  
Eugene Y. H. Yeung ◽  
Ivan Gorn
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Electrophoresis ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Epidemiological Studies ◽  
Pelvic Trauma ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Strains ◽  
Methicillin Resistant ◽  
Pulsed Field ◽  
Clinical Cases

Pulsed-field gel electrophoresis (PFGE) has historically been considered the gold standard in fingerprinting bacterial strains in epidemiological studies and outbreak investigations; little is known regarding its use in individual clinical cases. The current study detailed two clinical cases in which PFGE helped to determine the source of their methicillin-resistant Staphylococcus aureus (MRSA) bacteremia. Patient A was found to have MRSA bacteremia after trauma in her pelvic area. MRSA was also found in her groin but not in her nostril and rectum. PFGE was performed that showed variable bands of her MRSA isolates from blood and groin, suggestive of different strains of MRSA. Her MRSA bacteremia was determined to be unrelated to her pelvic trauma. Patient B was found to have MRSA bacteremia after colonoscopy. MRSA was also found in his nostril and rectum. PFGE was performed that showed variable bands of his MRSA isolates from blood and rectum but identical bands of MRSA isolates from his blood and nostril. His MRSA bacteremia was determined to be unrelated to his colonoscopy procedure. The current study demonstrates the use of PFGE to rule out the source of bacteremia in individual clinical cases.

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